Tyrosine kinase inhibitors and methods of using the same

ABSTRACT

Tyrosine kinase inhibitors and pharmaceutical composition comprising the same are disclosed. Methods of treating mammals who have p185 tumors and methods of preventing p185 tumors in mammals at elevated risks of developing such tumors are disclosed. The methods comprise the step of administering effective amounts pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent, and, a tyrosine kinase inhibitor to the mammal.

This application is a 371 of PCT/US00/03341 filed Feb. 9, 2000 whichclaim benefit of No. 60/119526 filed Feb. 10, 1999.

FIELD OF THE INVENTION

The present invention is related to tyrosine kinase inhibitors,pharmaceutical compositions that comprise the same, and methods of usingtyrosine kinase inhibitors to inhibit elevated tyrosine kinase activityassociated with tumors that express p185. The present invention relatesto methods of treating individuals who have cancer characterized bytumors with cells that express p185.

BACKGROUND OF THE INVENTION

The erbB family of receptors includes erbB1 (EGFR), erbB2 (p185), erbB3and erbG4. Ullrich, et al. (1984) Nature 309,418-425, which isincorporated herein by reference, describes EGFR. Schechter, A. L., etal. (1984) Nature 312, 513-516, and Yamamoto, T., et al.(1986) Nature319, 230-234, which are each incorporated herein by reference, describep185neu/erbB2. Kraus, M. H., et al. (1989) Proc. Natl. Acad. Sci. USA86, 9193-9197 which is incorporated herein by reference, describes erbB3Plowman, G. D., (1993) Proc. Natl. Acad. Sci. USA 90, 1746-1750, whichis incorporated herein by reference, describes erbB4.

The rat cellular protooncogene c-neu and its human counterpart c-erbB2encode 185 kDa transmembrane glycoproteins termed p185. Tyrosine kinase(tk) activity has been linked to expression of the transformingphenotype of oncogenic p185 (Bargmann et al., Proc. Natl. Acad. Sci.USA, 1988, 85, 5394; and Stem et al., Mol. Cell. Biol., 1988, 8, 3969,each of which is incorporated herein by reference). Oncogenic neu wasinitially identified in rat neuroglioblastomas (Schechter et al.,Nature, 1984, 312, 513, which is incorporated herein by reference) andwas found to be activated by a carcinogen-induced point mutationgenerating a single amino acid substitution, a Val to Glu substitutionat position 664, in the transmembrane region of the transforming protein(Bargmann et al., Cell, 1986, 45, 649, which is incorporated herein byreference). This alteration results in constitutive activity of itsintrinsic kinase and in malignant transformation of cells (Bargmann etal., EMBO J., 1988, 7, 2043, which is incorporated herein by reference).The activation of the oncogenic p185 protein tyrosine kinase appears tobe related to a shift in the molecular equilibrium from monomeric todimeric forms (Weiner et al., Nature, 1989, 339, 230, which isincorporated herein by reference).

Overexpression of c-neu or c-erbB2 to levels 100-fold higher than normal(i.e., >10⁶ receptors/cell) also results in the transformation of NIH3T3cells (Chazin et al., Oncogene, 1992, 7, 1859; DiFiore et al., Science,1987, 237, 178; and DiMarco et al., Mol. Cell. Biol., 1990, 10, 3247,each of which is incorporated herein by reference). However, NIH3T3cells or NR6 cells which express cellular p185 at the level of 10⁵receptors/cell are not transformed (Hung et al., Proc. Natl. Acad. Sci.USA, 1989, 86, 2545; and Kokai et al., Cell, 1989, 58, 287, each ofwhich is incorporated herein by reference), unless co-expressed withepidermal growth factor receptor (EGFR), a homologous tyrosine kinase(Kokai et al., Cell, 1989, 58, 287, which is incorporated herein byreference). Thus, cellular p185 and oncogenic p185 may both result inthe transformation of cells.

Cellular p185 is highly homologous with EGFR (Schechter et al., Nature,1984, 312, 513; and Yarnamoto et al., Nature, 1986, 319, 230, each ofwhich is incorporated herein by reference) but nonetheless is distinct.Numerous studies indicate that EGFR and cellular p185 are able tointeract (Stern et al., Mol. Cell. Biol., 1988, 8, 3969; King et al.,EMBO J, 1988, 7, 1647; Kokai et al., Proc. Natl. Acad. Sci. USA, 1988,85, 5389; and Dougall et al., J. Cell. Biochem., 1993, 53, 61; each ofwhich is incorporated herein by reference). The intermolecularassociation of EGFR and cellular p185 appear to up-regulate EGFRfunction (Wada et al., Cell, 1990, 61, 1339, which is incorporatedherein by reference). In addition, heterodimers which form active kinasecomplexes both in vivo and in vitro can be detected (Qian et al., Proc.Natl Acad Sci. USA. 1992, 89, 1330, which is incorporated herein byreference).

Similarly, p 185 interactions with other erbB family members have beenreported (Carraway et al., Cell 1994, 78, 5-8; Alroy et al., FEBS Lett.1997, 410, 83-86; Riese et al., Mol. Cell. Biol. 1995, 15,5770-5776;Tzahar et al., EMBO J, 1997, 16, 4938-4950; Surden et al.,Neuron 1997, 18, 847-855; Pirlcas-Kramarski et al., Oncogene 1997, 15,2803-2815; each of which is incorporated herein by reference). Humanp185 forms heterodimers with either erbB3 or erbB4 under physiologicconditions, primarily in cardiac muscle and the nervous system,particularly in development.

Cellular p185 proteins are found in adult secretory epithelial cells ofthe lung. salivary gland, breast, pancreas, ovary, gastrointestinaltract, and skin (Kokal et al., Proc. Natl. Acad. Sci. USA, 1987, 84,8498; Mori et al., Lab, Invest., 1989, 61, 93; and Press et al.,Oncogene, 1990, 5, 953; each of which is incorporated herein byreference). Recent studies have found that the amplification of c-erbB2occurs with high frequency in a number of human adenocarcinomas such asgastric (Akivama et al., Science, 1986, 232, 1644, which is incorporatedherein by reference), lung (Kern et al., Cancer Res., 1990, 50, 5184,which is incorporated herein by reference) and pancreaticadenocarcinomas (Williams et al., Pathobiol., 1991, 59, 46, which isincorporated herein by reference). It has also been reported thatincreased c-erbB2 expression in a subset of breast and ovariancarcinomas is linked to a less optimistic clinical prognosis (Slamon etal., Science, 1987, 235, 177; and Slamon et al., Science, 1989, 244,707, each of which is incorporated herein by reference). Heterodimericassociation of EGFR and p185 has also been detected in human breastcancer cell lines, such as SK-Br-3 (Goldman et al., Biochemistry, 1990,29, 11024, which is incorporated herein by reference), and transfectedcells (Spivak-Kroizman et al., J. Biol. Chem., 1992, 267, 8056, which isincorporated herein by reference). Additionally, cases of erbB2 and EGFRcoexpression in cancers of the breast and prostate have been reported.In addition, heterodimeric association of p185 and erbB3 as well asheterodimeric association of p185 and erbB4 have also been detected inhuman cancers. Coexpression of erbB2 and erbB3 has been observed inhuman breast cancers. Coexpression of EGFR, erbB2, and erbB3 has beenseen in prostate carcinoma.

As used herein, the term “p185” is meant to refer to the neu geneproduct and the erbB-2 gene product which are 185 kdalton receptorproteins as determined by carrying out electrophoresis on theglycoprotein and comparing its movement with marker proteins of knownmolecular weight. p185 has tyrosine kinases activity, forms homodimerswith themselves and interacts with other members of the erbB family,such as erbB1 (epidermal growth factor receptor or EGFR), erbB3 anderbB4 to form heterodimers.

As used herein, the term “p185 tumors”, “neu-associated cancer”,“neu-associated tumors” and “p185-associated tumors” are usedinterchangably and are meant to refer to tumors with cells that expressp185. A p185 tumor may contain cells that express p185 and,additionally, other members of the erbB family, such as erbB1 (EGFR),erbB3 and erbB4 to form heterodimers and EGFR. A p185 tumor may containp185 homodimers and/or heterodimers including p185-EGFR heterodimersand/or p185-erbB3 heterodimers and/or p185-erbB4 heterodimers.Dimerization of p185 with other p185 molecules or other members of thep185 family is associated with elevated tyrosine kinase activity.Examples of p185 tumors include many human adenocarcinomas such as somebreast, ovary, lung, pancreas, salivary gland, kidney, prostateadenocarcinomas and some neuroblastoma.

As used herein, the term “p185-mediated cellular transformation” ismeant to refer to the cellular transformation that p185-associated tumorcell neoplasms undergo and whose transformed phenotype can be arrestedand/or reversed by tyrosine kinase inhibitors.

As used herein, the term “high risk individual” is meant to refer to anindividual who either a) has had a p185-associated tumor either removedor enter remission and who is therefore susceptible to a relapse orrecurrence or b) has a genetic predisposition to develop p185 tumors.Individual who have had p185-associated tumor either removed or enterremission can be readily identified through personal medical history.Individuals who have a genetic predisposition to develop p185 tumors canbe identified by those skilled in the art using various means such as byreview of family medical history. As part of a treatment regimen for ahigh risk individual, the individual can be prophylactically treatedagainst tumors that they have been diagnosed as having had in order tocombat a recurrence or tumor development. Thus, once it is known that anindividual has had a p185-associated cancer, the individual can betreated according to the present invention to prevent normal cells fromtransforming into tumor cells.

As used herein, the term “therapeutically effective amount” is meant torefer to an amount of a compound which produces a medicinal effectobserved as reduction or reverse in tumorigenic phenotype of tumor cellsin an individual when a therapeutically effective amount of a compoundis administered to an individual who is susceptible to or suffering fromp185 tumors. Therapeutically effective amounts are typically determinedby the effect they have compared to the effect observed when acomposition which includes no active ingredient is administered to asimilarly situated individual.

As used herein, the term “prophylactically effective amount” is meant torefer to an amount of a compound which produces a medicinal effectobserved as the prevention of non-transformed cells from becomingtransformed in an individual when a prophylactically effective amount ofa compound is administered to an individual who is susceptible to p185tumors. Prophylactically effective amounts are typically determined bythe effect they have compared to the effect observed when a compositionwhich includes no active ingredient is administered to a similarlysituated individual.

The present invention is useful to therapeutically treat an individualidentified as suffering from p185-associated tumors in order to reversethe transformed phenotype of the tumor cells. The present invention isuseful to prophylactically treat an individual who is predisposed todevelop p185-associated tumors or who has had p185-associated tumors andis therefore susceptible to a relapse or recurrence.

The present invention provides novel compounds which have tyrosinekinase inhibitor activity and a formula selected from the groupconsisting of Formula 1, Formula 2, Formula 3 and Formula 4, as setforth in the section below entitled Formulae, or a pharmaceuticallyacceptable salt thereof.

The invention provides novel pharmaceutical compositions comprisingtyrosine kinase inhibitors which can reduce the enhanced tyrosine kinaseactivity associated with p185 homodimers and p185-EGFR heterodimers,p185-erbB3 heterodimers and p185-erbB4 heterodimers. The tyrosine kinaseinhibitors included in the pharmacuetical compositions of the presentinvention have a formula selected from the group consisting of Formula1, Formula 2, Formula 3 and Formula 4, as set forth in the section belowentitled Formulae, or a pharmaceutically acceptable salt thereof.

In some preferred embodiments, the tyrosine kinase inhibitor in thepharmacuetical compositions of the present invention has a formula ofFormula 1 as set forth in the section below entitled Formulae. In somepreferred embodiments, the tyrosine kinase inhibitor in thepharmacuetical compositions of the present invention has a formulaof-Formula 1 COMP11 (F₁ ¹), COMP12 (F₂ ¹), COMP13 (F₃ ¹), COMP14 (F₄ ¹),COMP15 (F₅ ¹), or COMP16 (F₆ ¹), as set forth in the section belowentitled Formulae, or a pharmaceutically acceptable salt thereof.

In some preferred embodiments, the tyrosine kinase inhibitor in thepharmacuetical compositions of the present invention has a formula ofFormula 2 as set forth in the section below entitled Formulae. In somepreferred embodiments, the tyrosine kinase inhibitor in thepharmacuetical compositions of the present invention has a formula ofFormula 2 COMP21 (F₁ ²), COMP22 (F₂ ²), COMP23 (F₃ ²), COMP24 (F₄ ²), orCOMP 25(F₅ ²), as set forth in the section below entitled Formulae, or apharmaceutically acceptable salt thereof.

In some preferred embodiments, the tyrosine kinase inhibitor in thepharmacuetical compositions of the present invention has a formula ofFormula 3 as set forth in the section below entitled Formulae. In somepreferred embodiments, the tyrosine kinase inhibitor in thepharmacuetical compositions of the present invention has a formula ofFormula 3 COMP31 (F₁ ³), COMP32 (F₂ ³), COMP33 (F₃ ³), or COMP34 (F₄ ³),as set forth in the section below entitled Formulae. In some preferredembodiments, the tyrosine kinase inhibitor in the pharmacueticalcompositions of the present invention has a formula of Formula 4 as setforth in the section below entitled Formulae. In some preferredembodiments, the tyrosine kinase inhibitor in the pharmacueticalcompositions of the present invention has a formula of Formula 4 COMP41(F₁ ⁴), or COMP42 (F₂ ⁴), as set forth in the section below entitledFormulae, or a pharmaceutically acceptable salt thereof.

The invention provides methods for treating mammals who have p185tumors. A tyrosine inhibitor according to Formula 1, Formula 2, Formula3 or Formula 4 is administered to the mammal in an amount effective tosuppress tumor growth. The tyrosine kinase inhibitor reduces tyrosinekinase activity and thus suppresses tumor growth. In preferredembodiments, the tyrosine kinase inhibitor is COMP11(F₁ ¹), COMP12 (F₂¹), COMP13 (F₃ ¹), COMP14 (F₄ ¹) COMP15 (F₅ ¹), COMP16 (F₆ ¹), COMP21(F₁ ²), COMP22 (F₂ ²), COMP23 (F₃ ²), COMP24 (F₄ ²), COMP25 (F₅ ²),COMP31 (F₁ ³), COMP32 (F ₂ ³), COMP33 (F₃ ³), COMP34 (F₄ ³), COMP41 (F₁⁴); or COMP42 (F₂ ⁴), . . . as set forth in the section below entitledFormulae, or a pharmaceutically acceptable salt thereof.

The invention also provides novel therapeutic compositions for treatingmammals who have p185 tumors. The methods comprise admininstering tosuch mammals a therapetucially effective amount of a tyrosine kinaseinhibitor according to Formula 1, Formula 2, Formula 3 or Formula 4. Inpreferred embodiments, the tyrosine kinase inhibitor is COMP11 (F₁ ¹),COMP12 (F₂ ¹), COMP13 (F₃ ¹), COMP14 (F₄ ¹), COMP15 (F₅ ¹), COMP16 (F₆¹), COMP21 (F₁ ²), COMP22 (F₂ ²), COMP23 (F₃ ²),COMP24 (F₄ ²), COMP25(F₅ ²), COMP31 (F₁ ³), COMP32 (F₂ ³), COMP33 (F₃ ³), COMP34 (F₄ ³),COMP41 (F₁ ⁴), or COMP42 (F₂ ⁴), as set forth in the section belowentitled Formulae, or a pharmaceutically acceptable salt thereof.

COMP11 (F₁ ¹), COMP 12 (F₂ ¹), COMP13 (F₃ ¹), COMP14 (F₄ ¹), COMP15 (F₅¹), COMP16 (F₆ ¹), COMP21 (F₁ ²), COMP22 (F₂ ²), COMP23 (F₃ ²), COMP24(F₄ ²), COMP25 (F₅ ²), COMP31 (F₁ ³), COMP32 (F₂ ³), COMP33 (F₃ ³),COMP34 (F₄ ³), COMP41 (F₁ ⁴), or COMP42 (F₂ ⁴), are each commerciallyavailable chemical entities as set forth in the section below entitledSources.

In some embodiments, the method of the invention additionally includesthe use of the tyrosine kinase inhibitors in combination with othermethodologies to treat tumors. In some embodiments, the tyrosine kinaseinhibitor is administered in conjunction with other chemotherapeuticagents. In some embodiments, the tyrosine kinase inhibitor isadministered in conjunction with radiation therapy. In some embodiments,the tyrosine kinase inhibitor is administered in conjunction with otherchemotherapeutic agents and radiation therapy.

In some embodiments of the invention, the expression of p 185 by cellsof the tumor are identified prior to administration of the tyrosinekinase inhibitor. In some embodiments, samples of tumors may be removedsuch as biopsy samples and tested to identify the presence of p185 usingp185-specific antibodies. The antibodies are preferably monoclonalantibodies. Those having ordinary skill in the art can routinelygenerate antibodies specific for p185 such as by the method in Harlowand Lane, eds., Antibodies: A Laboratory, Manual. Cold Spring HarborLaboratory. Cold Spring Harbor. N.Y. 1988. A general method for theproduction of monoclonal antibodies comprises the steps of immunizing ananimal such as a mouse or rat with an antigen to which monoclonalantibodies are desired. After allowing time for the immune system togenerate lymphocytes capable of producing antibodies to the antigen, theanimal is sacrificed and a suspension of spleen cells is prepared. Thespleen cells are then fused with myeloma cells by contacting them in thepresence of a fusion promoter such as polyethylene glycol. A percentageof the cells fuse to produce hybridomas. The earlier immunization of theanimal from which the spleen cells were removed results in a number oflymphocytes which secrete antibody to the antigen of interest, acharacteristic that is transferred genetically to the hybridoma duringfusion of the spleen and myeloma cells. Hybridomas secreting monoclonalantibody having the desired specificity are then isolated using routinescreening techniques. Antibodies against p185 are described in U.S. Pat.No. 5,677,171 issued Oct. 14, 1997, which is incorporated herein byreference, and U.S. Pat. No. 5,705,157 issued Jan. 6, 1998, which isincorporated herein by reference. Peptidomimetics of antibodies againstp185 which bind to p185 and which can be used to identify the presenceof p185 are described in U.S. Pat. No. 5,663,144 issued Sep. 2, 1997,which is incorporated herein by reference. Other methods for identifyingtumors which express p185 may be designed routinely.

The pharmaceutical compositions comprising tyrosine kinase inhibitors ofthe present invention may be administered by any means that enables theactive agent to reach the agent's site of action in the body of amammal. Pharmaceutical compositions of the present invention may beadministered by conventional routes of pharmaceutical administration.Pharmaceutical compositions may be administered parenterally, i.e.,intratumor, intravenous, subcutaneous, intramuscular. Intravenous andintratumor administration are preferred routes. In some embodiments, thepharmaceutical compositions are administered orally. Pharmaceuticalcompositions are administered to the mammal for a length of timeeffective to reduce tumor size and as needed to maintain regression ofthe tumor.

Pharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

Dosage varies depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. Usually a daily dosage of activeingredient can be about 0.001 to 1 grams per kilogram of body weight, insome embodiments about 0.1 to 100 milligrams per kilogram of bodyweight. Ordinarily dosages are in the range of 0.5 to 50 milligrams perkilogram of body weight, and preferably 1 to 10 milligrams per kilogramper day. In some embodiments, the pharmaceutical compositions are givenin divided doses 1 to 6 times a day or in sustained release form iseffective to obtain desired results.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95 by weight based on the total weight of the composition.

Subsequent to initial administration, individuals may be boosted byreadministration. In some preferred embodiments, multipleadministrations are performed.

Pharmaceutical compositions may be formulated by one having ordinaryskill in the art with compositions selected depending upon the chosenmode of administration. Suitable pharmaceutical carriers are describedin Remingion's Pharmaceutical Sciences, A. Osol, a standard referencetext in this field, which is incorporated herein by reference.

For parenteral administration, the compound can be formulated as asolution, suspension, emulsion or lyophilized powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Liposomes and nonaqueous vehicles such as fixedoils may also be used. The vehicle or lyophilized powder may containadditives that maintain isotonicity (e.g., sodium chloride mannitol) andchemical stability (e.g. buffers and preservatives). The formulation issterilized by commonly used techniques. In some embodiments, aparenteral composition suitable for administration by injection isprepared by dissolving 1.5% by weight of active ingredient in 0.9%sodium chloride solution.

According to some embodiments of the present invention, the compositionis administered to tissue of an individual by topically or by lavage.The compounds may be formulated as a cream, ointment, salve, douche,suppository or solution for topical administration or irrigation.Formulations for such routes administration of pharmaceuticalcompositions are well known. Generally, additives for isotonicity caninclude sodium chloride, dextrose. mannitol, sorbitol and lactose.

In some cases, isotonic solutions such as phosphate buffered saline areused. Stabilizers include gelatin and albumin. In some embodiments, avasoconstriction agent is added to the formulation. The pharmaceuticalpreparations according to the present invention are preferably providedsterile and pyrogen free.

A pharmaceutically acceptable formulation will provide the activeingredient(s) in proper physical form together with such excipients,diluents, stabilizers, preservatives and other ingredients as areappropriate to the nature and composition of the dosage form and theproperties of the drug ingredient(s) in the formulation environment anddrug delivery system.

In some embodiments, the invention relates to methods of treatingpatients suffering from human adenocarcinomas which are p185-associatedcancers such as gastric, lung and pancreatic adenocarcinomas and humanbreast and ovarian carcinomas as well as human breast and prostatecancer which are p185-associated cancer. In some embodiments, theinvention relates to methods of preventing these p185-associated cancersin high risk individuals. In some embodiments, the invention relates tomethods of preventing these p185-associated cancers in high riskindividuals.

In some embodiments, the invention relates to methods of treatingpatients suffering from human epithelial malignancies erythroidleukemia, fibrosarcoma, angiosarcoma and melanoma. In some embodiments,the invention relates to methods of preventing these p185-associatedcancers in high risk individuals.

According to some embodiments of the invention, the patient is treatedwith radiation or other chemotherapy in conjunction the administrationof pharmaceutical compositions according to the invention. The use ofmultiple therapeutic approaches provides the patient with a broaderbased intervention.

According to some aspects of the present invention, in combination withadministration of the composition that comprises the tyrosine kinaseinhibitor, the individual is then administered a cytotoxicchemotherapeutic agent. In some embodiments, in combination withadministration of the composition that comprises the tyrosine kinaseinhibitor, the individual is exposed to chemotherapeutic agents and,additionally, to a therapeutic amount of gamma radiation. Chemotherapyapproaches include administration of cytotoxic and or cytostatic agents.Chemotherapeutics are delivered according to standard protocols usingstandard agents, dosages and regimens. In some embodiments, thechemotherapeutic is selected from the group consisting of: cisplatin,doxirubicin, danurubicin, tamoxiphen, taxol, and methotrexate. Otherexamples of chemotherapeutics useful in the present invetion include,but are not limited to: cytosinarabinoside, etoposide, 5-4 fluorouracil,melphalan, chlorambucil, and other nitrogen mustards (e.g.cyclophosphamide), vindesine (and other vinca alkaloids), mitomycin andbleomycin. As a step in the method of the invention, chemotherapeuticsmay be administered to patients being treated or who have treated fortumors that express p185.

In some embodiments, radiotherapy follows administration ofpharmaceutical compositions according to the invention. In somepreferred embodiments, the radiation therapy using gamma radiation isprovided following administration of compositions which convertradiation resistant tumors, radiation sensitive. According to aspects ofthe present invention, after administering the composition thatcomprises the tyrosine kinase inhibotir, the individual is then exposedto a therapeutic amount of gamma radiation. Gamma radiation is deliveredaccording to standard radiotherapeutic protocols using standard dosagesand regimens. Those skilled in the art can readily formulate anappropriate radiotherapeutic regimen. Carlos A Perez & Luther W Brady:Principles and Practice of Radiation Oncology, 2nd Ed. JB Lippincott Co,Phila., 1992, which is incorporated herein by reference describesradiation therapy protocols and parameters which can be used in thepresent invention. For GBMs (glioblastoma, the most malignant glialbrain tumor). Simpson W. J. et al., Influence of location and extent ofsurgical resection on survival of patients with glioblastoma multiforms:Results of three consecutive Radiation Therapy Oncology Group (RTOG)clinical trials. Int J Radiat Oncol Biol Phys 26:239-244, 1993, which isincorporated herein by reference describes clinical protocols useful inthe methods of the present invention. Similarly, for Borgelt et al., Thepalliation of brain metastases: Final results of the first two studiesof the Radiation Therapy Oncology Group Int J Radial Oncol Biol Phys6:1-9, 1980, which is incorporated herein by reference, describesclinical protocols useful in the methods of the present invention.

In some preferred embodiments, treatment with pharmaceuticalcompositions according to the invention is preceded by surgicalintervention. According to some preferred embodiments, the presentinvention provides anti-cancer gene therapy treatment to treat residual,local disease, as a therapeutic adjuvant in combination with preexistingtreatments. Delivery is local at the time of surgery, most likely afterthe resection of all gross disease. According to some embodiments of theinvention, the pharmaceutical compositions are administered locally atthe site of the tumor. In some embodiments, the pharmaceuticalcompositions are administered directly into the tumor cells and thetissue immediately surrounding the tumor.

In addition to treating mammals with p185 tumors, the present inventionrelates to methods of preventing tumors in any patient populationidentified as being susceptible to p185 tumors, it is particularlyuseful in high risk individuals who, for example, have a family historyof cancer characterized by such tumors or those who show a geneticpredisposition. Additionally, the present invention is particularlyuseful to prevent recurrence of such tumors in patients who have hadsuch tumors removed by surgical resection or who have been diagnosed ashaving such cancer in remission.

Those having ordinary skill in the art can readily identify individualswho are susceptible to such tumors, particularly those individualsconsidered to be a high risk for whom the methods of the invention areparticularly useful. Advancements in the understanding of genetics anddevelopments in technology as well as epidemiology allow for thedetermination of probability and risk assessment an individual has fordeveloping cancer. Using family health histories and/or geneticscreening, it is possible to estimate the probability that a particularindividual has for developing certain types of cancer. Those individualsthat have been identified as being predisposed to developing aparticular form of cancer can take only limited prophylactic stepstowards reducing the risk of cancer. There is no currently availablemethod or composition which can chemically intervene with thedevelopment of cancer and reduce the probability a high risk individualwill develop cancer. Similarly, those individuals who have alreadydeveloped cancer and who have been treated to remove the cancer or areotherwise in remission are particularly susceptible to relapse andreoccurrence.

In preferred embodiments, the methods of the present invention areuseful to prophylactically and therapeutically treat humans.

The present invention is not intended to be limited by any theory. Thepresent invention is further illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE Example 1 Tyrosine Kinase Activity

Membrane Purification

Cells were lysed by a combination of snap freeze-thawing and Douncehomogenization as described in Gaulton et al., J Immunol., 1986, 7,2470, which is incorporated herein by reference. The nuclear fractionwas removed by centrifugation at 2000 ×g for 5 minutes. The 2000 ×gsupernatant fraction was then recentrifuged at 25000 ×g for 30 minutesat 4° C., and the 25000 ×g supernatant was retained as the cytosolfraction. The pellet was redissolved in 1.5 ml of membrane buffer (40 mMNaCl, 0.1 mM EDTA, 20 mM HEPES (pH 6.8), 2 mM PMSF, and 5 mM Napyrophosphate) then layered over a (20%-37%) sucrose solution inmembrane buffer and centrifuged at 22000 rpm for 18 hours at 2° C byusing a Beckrnan SW50.1 rotor. The membrane-rich interface was removedin 1 ml total volume, diluted with 10 ml of membrane buffer, and wasrecentrifuged at 40000 rpm for 60 minutes by using an SW40.1 rotorexactly as described in Zick et al., Biochem. Biophys. Res. Commun.,1984, 119, 6, which is incorporated herein by reference. The resultantpellet containing purified membrane fragments, was redissolved in 100 μlof Kinase buffer (see below) per 10⁷ original cells. Membrane proteinswere quantitated using a BioRad protein assay kit and stored at −80° C.until assay,

Tyrosine Kinase Actvity In Membranes

Membrane concentrations were determined by the method of Bradford asdescribed in Gaulton et al., J Immunol., 1986 7, 2470, which isincorporated herein by reference. Dilutions of membranes were incubatedin quadruplicate in the presence or absence of synthetic polypeptidecontaining tyrosine as a specific indicator of tyrosine phosphorylation.Kinase reaction buffer, (50 μl of 0.1 M Hepes pH 7.3, 10 mM MgCl₂, 5 mMMnCl², 50 μM Na₃VO₄ were incubated in the presence of ATP (1 μCi ofgamma [³²P]ATP; Amersham) for 5 minutes at room temperature. Reactionswere halted by adding 5 mM EDTA (final concentration) followedimmediately by TCA immunoprecipitation onto glass fiber filters (WhatmanGF/A). Filters were washed extensively with TCA followed by ether,air-dried, immersed in scintillation cocktail (Biofluor) and betaemissions determined. Quadruplicate wells assayed in the absence oftyrosine containing substrate were subtracted from tyrosine substratecontaining wells.

Membrane proteins were incubated with the random polymer of glutamicacid-tyrosine (4:1) poly glu:tyr, PGT) as substrate for tyrosinephosphorylation as described in Zick et al., Biochem. Biophys. Res.Commun., 1984, 119, 6, which is incorporated herein by reference.Briefly, membrane proteins were incubated in 50 μl of 10 mM HEPES pH7.2containing 10 mM MgCl₂, 100 μM Na₃VO₄and 150 μM (10 μCi) [³²P]ATP for15 minutes at room temperature in the presence (specific) or absence(background) of poly glu:tyr substrate at 2.5 mg/ml. Reactions werestopped by the addition of EDTA to 50 mM final concentration and coldexcess ATP and samples were spotted onto Whatman glass fiber filterpaper. Filters were washed 3 times with ice cold 10% TCA containing 10mM pyrophosphate and 1 mM ATP followed by once with acetate. Sampleswere then dried and counted in BioFlur (NEN). For immunoprecipitation ofphosphotyrosine containing membrane proteins, 50 μg of purifiedmembranes were incubated in kinase buffer as described above for 15minutes. After labeling, samples were solubilized in Lysis buffersupplemented with 5 mM EDTA, precleared and immune precipitated with 2μl ascites from MA-2G8A6 +protein A agarose. The MA-2G8 antibodyspecifically precipitates phosphotyrosine labeled polypeptides asdescribed in Daniel et al., Proc. Natl. Acad. Sci. USA, 1985, 82, 2084,which is incorporated herein by reference.

SOURCES Compound Source Company Catalog Number 11 - F₁ ¹ MAYBRIDGE PD00154 12 - F₂ ¹ G&J RES JS241 13 - F₃ ¹ G&J JS258 14 - F₄ ¹SALOR/ALDRICH S11.804-4 15 - F₅ ¹ SALOR/ALDRICH S19,987-7 16 - F₆ ¹SALOR/ALDRICH 23,838-4 21 - F₁ ¹ APIN-NP N069511 22 - F₂ ² APIN-NPN11493C 23 - F₃ ² KNOLL 00105 24 - F₄ ² FLUKA 91950 25 - F₅ ² SPECSCIF6409 31 - F₁ ³ MAYBRIDGE SEW 04598 32 - F₂ ³ MAYBRIDGE SEW 04759 33 -F₃ ³ MAYBRIDGE DP 01012 41 - F₁ ⁴ SALOR/ALDRICH S25,931-4 42 - F₂ ⁴ G&JRESEARCH JS572

List Of Company Addresses

Aldrich Chemical Company, Inc.

1001 West Saint Paul Avenue

Milwaukee, Wis. 53233

USA

Fluka Chemie AG

Industriestrasse 25

P.O. Box 260

CH-9470 Buchs

SWITZERLAND

Apin Chemical Ltd.

Unit 29D

Milton Park

Near Abingdon

Oxon OX 14 4RT

UNITED KINGDOM

G&J Research Chemicals Ltd.

Fernworthy

Bridestowe

Okehampton, Devon, UK

Knoll AG

Postfach 21 08 05

D-67008 Ludwigschafen

GERMANY

Maybridge Chemical Company Ltd.

Trevillett

Tintagel

Comwall PL34 OHW

UK

SPECS and BioSPECS B.V.

Fleminglaan 16

2289 CP Rljswljk

THE NETHERLANDS

Formulae

What is claimed is:
 1. A method of treating a mammal who has a p185tumor, said method comprising the step of administering to said mammalan amount of a pharmaceutical composition effective to reduce tumorgrowth, the pharmaceutical composition comprising: (a) a compound havingthe structure

or a pharmaceutically acceptable salt thereof, wherein R₁ is

(b) a pharmaceutically acceptable carrier or diluent.
 2. The method ofclaim 1 wherein said pharmaceutical composition is administered incombination with a therapeutically effective amount of anti-cancerradiation.
 3. The method of claim 1 wherein said pharmaceuticalcomposition is administered in combination with one or more otheranti-tumor compositions.
 4. The method of claim 3 wherein said otheranti-tumor compositions are selected from the group consisting of:cisplatin, doxirubicin, danurubicin, tamoxiphen, taxol, methotrexate,cytosinarbinoside, etoposide, 5-4 fluorouracil, melphalan, chlorambucil,nitrogen mustards, vindesine, mitomycin and belomycin.
 5. A methodaccording to claim 1, wherein the pharmaceutical composition comprises acompound having the structure:

or a pharmaceutically acceptable salt thereof.
 6. A method according toclaim 1, wherein the pharmaceutical composition comprises a compoundhaving the structure:

or a pharmaceutically acceptable salt thereof.